Performance of three generations of Xpert MRSA in routine practice: approaching the aim?

The aim of this study was to evaluate retrospectively the performance of the Xpert MRSA assay in routine practice and its current use in the intensive care unit (ICU) setting of our hospital, since a pre-emptive isolation strategy has been applied. A total of 6473 patients were routinely screened with ESwab for methicillin-resistant Staphylococcus aureus (MRSA) using three generations of rapid real-time polymerase chain reaction (PCR) (Cepheid GeneXpert) over three consecutive periods of time. Performance was evaluated using broth enrichment culture as the reference method. Our results show that the last generation of Xpert MRSA (NxG) assay is more specific (99.2% vs. 97.9%) but not more sensitive (77.8% vs. 86.9%) than the third generation. Considering the low prevalence of MRSA in our hospital, we obtained an overall low positive predictive value. In conclusion, it remains difficult to abandon the reference method in routine practice considering the possible implications of an erroneous MRSA result in the ICU.

How is post-mortem microbiology appraised by pathologists? Results from a practice survey conducted by ESGFOR.

Post-mortem microbiology (PMM) is an important tool in forensic pathology, assisting to determine the cause and manner of death. However, there is a lack of standardisation of PMM sampling. In order to get a better insight into the methods used, the available technical options and developmental needs, ESCMID Study Group for Forensic and Postmortem Microbiology (ESGFOR) members designed a survey aimed at pathologists regarding common practices of PMM in clinical and forensic autopsies. Multiple choice questions were developed based on Cumulative Techniques and Procedures in Clinical Microbiology (Cumitech). The questionnaire was sent to pathologists mainly across Europe and Turkey using SurveyMonkey. The survey had 147 respondents. Although all pathologists were aware of the existence of PMM, 39% (19/49) of the participants were not using it. The three main indications for PMM were: (i) clinical suspicion of an infection not confirmed antemortem (83%), (ii) infectious signs at autopsy (83%) and (iii) as part of a standard protocol for foetal/perinatal or paediatric death (67%). Almost 80% of the participants using PMM stated taking 1-10 samples per case. Of the requested examinations, a general bacteriological culture (96%) and a specific polymerase chain reaction (PCR) assay for a particular infectious agent (34%) were most popular. The most frequent samples were: heart blood (66%), peripheral femoral blood (49%), spleen (64%) and lung (56%). Eighty-nine percent of the participants considered PMM a useful resource when investigating the cause of death. Although there are some common uses, this survey indicates that there is a need for improvement towards standardising sampling procedures in PMM.

Technical and clinical validation of three commercial real-time PCR kits for the diagnosis of neuroborreliosis in cerebrospinal fluid on three different real-time PCR platforms.

This study reports the evaluation of the technical and clinical validation of the O-DiaBorburg kit (DIA), Borrelia burgdorferi PCR kit, ISEX (GENE), and Borrelia burgdorferi sensu lato Real-TM (SAC) for the diagnosis of neuroborreliosis in cerebrospinal fluid based on both Borrelia DNA and CSF samples from patients with clinical suspicion of neuroborreliosis. This validation study was done by analysing the kits on the Rotorgene Q (RGQ), CFX96, and LightCycler480 (LC480). For all kits, the linear range was larger on RGQ than on CFX96 and LC480. A good reproducibility was obtained for all assays on all instruments. Storage at -20 °C resulted in a decreased reproducibility for SAC. Results of the limit of detection (LOD95) experiments indicated a better sensitivity than described in the kit insert for all kits on all PCR platforms. No cross-reactivity was found for genetically related organisms nor for other pathogens which may be present in CSF. All species of the Borrelia burgdorferi sensu lato complex were detected with the GENE and SAC kits. The DIA kit failed to detect B. lusitaniae. The results seemed to indicate a better overall performance for the GENE kit on RGQ. However, its diagnostic value could not be confirmed in the clinical validation study, wherein none of the 103 CSF samples from clinical neuroborreliosis cases showed a positive real-time PCR result with the GENE kit analysed on RGQ.

Typing of Pneumocystis jirovecii by multilocus sequencing: evidence of outbreak?

Different reports of Pneumocystis jirovecii pneumonia (PcP) outbreaks on oncology and transplant units suggest the possibility of a person-to-person transmission. Based on these reports, we searched retrospectively for possible PcP clusters in UZ Leuven in 2013. A movement and transmission map was established for all patients (n = 21) with a positive PcP PCR on BAL fluid. BAL fluid samples from all patients with a positive PCR on the mitochondrial large subunit mRNA of P. jirovecii and possible cross exposure were typed with multilocus sequence typing (MLST). Five patients with a positive PcP PCR could have contact with another PcP patient. Another five patients with a weak positive PcP PCR on BAL fluid during the same period were also included. Based on the MLST typing of the BAL samples of these ten patients, there was no evidence of a PcP outbreak in UZ Leuven in 2013. MLST has proven to be a useful tool in genotyping and outbreak detection. From this case series, it could be concluded that current infection control precautions for P. jirovecii are appropriate in UZ Leuven. However, there is need for an international Pneumocystis database and more clarity in the geographic distribution of different P. jirovecii genotypes.

Binary toxin and its clinical importance in Clostridium difficile infection, Belgium.

Binary toxin-producing Clostridium difficile strains such as ribotypes 027 and 078 have been associated with increased Clostridium difficile infection (CDI) severity. Our objective was to investigate the association between presence of the binary toxin gene and CDI severity and recurrence. We performed a laboratory-based retrospective study including patients between January 2013 and March 2015 whose fecal samples were analyzed by polymerase chain reaction (PCR) for the presence of the genes for toxin B and binary toxin and a deletion in the tcdC gene, specific for ribotype 027. Clinical and epidemiological characteristics were compared between 33 binary toxin-positive CDI patients and 33 binary toxin-negative CDI patients. Subsequently, the characteristics of 66 CDI patients were compared to those of 66 diarrhea patients who were carriers of non-toxigenic C. difficile strains. Fifty-nine of 1034 (5.7 %) fecal samples analyzed by PCR were binary toxin-positive, belonging to 33 different patients. No samples were positive for ribotype 027. Binary toxin-positive CDI patients did not differ from binary toxin-negative CDI patients in terms of disease recurrence, morbidity, or mortality, except for a higher peripheral leukocytosis in the binary toxin-positive group (16.30 × 109/L vs. 11.65 × 109/L; p = 0.02). The second part of our study showed that CDI patients had more severe disease, but not a higher 30-day mortality rate than diarrhea patients with a non-toxicogenic C. difficile strain. In our setting with a low prevalence of ribotype 027, the presence of the binary toxin gene is not associated with poor outcome.

'Bedside assessment' of acute hantavirus infections and their possible classification into the spectrum of haemophagocytic syndromes.

Hantavirus infections, recently renamed 'hantavirus fever' (HTVF), belong to the most common but also most underestimated zoonoses in the world. A small number of reports described the so-called 'lipid paradox' in HTVF, i.e. the striking contrast between a very low serum total cholesterol and/or high-density lipoprotein cholesterol (HDLc), and a paradoxical concomitant hypertriglyceridaemia. In a prospective study, with patients being their own control after illness, we wanted to verify if this quick and easy 'bedside test' was robust enough to warrant a preliminary diagnosis of acute kidney injury (AKI) caused by HTVF. The study cohort consisted of 58 Belgian cases (mean age 44 years), admitted with varying degrees of AKI and of thrombocytopaenia, both characteristic for presumptive HTVF. All cases were sero-confirmed as having acute HTVF. At or shortly after hospital admission, a significant (p < 0.001) decrease of total cholesterol and HDLc was found in comparison with normalised levels in the same cohort, quantified a few days after spontaneous AKI recovery. Conversely, fasting triglyceride levels during HTVF infection were significantly (p < 0.001) higher during illness than after recovery. This 'lipid paradox' was most outspoken in severe HTVF cases, often accompanying, or even predicting, major kidney or lung complications. Thus, this 'bedside assessment' seems to hold even promise for presumptive diagnosis of more severe so-called 'hantavirus cardio-pulmonary syndrome' (HCPS) cases, mostly described hitherto in the New World. In more severe AKI cases, the mean total cholesterol was significantly lower (p = 0.02) than in milder cases, i.e. cases with peak serum creatinine levels of < 1.5 mg/dL. Thrombocytopaenia, generally accepted as the severity index in HTVF, appeared, moreover, significantly correlated with serum levels of total cholesterol (R = 0.52, p < 0.001) and with serum levels of HDLc (R = 0.45, p < 0.01). A link with the novel clinical entity of haemophagocytic syndromes, also characterised by manifest hypertriglyceridaemia, is discussed.

Adherence to infection control guidelines in surgery on MRSA positive patients : A cost analysis.

In surgical units, similar to other healthcare departments, guidelines are used to curb transmission of methicillin resistant Staphylococcus aureus (MRSA). The aim of this study was to calculate the extra costs for material and extra working hours for compliance to MRSA infection control guidelines in the operating rooms of a University Hospital. The study was based on observations of surgeries on MRSA positive patients. The average cost per surgery was calculated utilizing local information on unit costs. Robustness of the calculations was evaluated with a sensitivity analysis. The total extra costs of adherence to MRSA infection control guidelines averaged €â€Š340.46 per surgical procedure (range €â€Š207.76- €â€Š473.15). A sensitivity analysis based on a standardized operating room hourly rate reached a cost of €â€Š366.22. The extra costs of adherence to infection control guidelines are considerable. To reduce costs, the logistical planning of surgeries could be improved by for instance a dedicated room.

How to optimise the yield of forensic and clinical post-mortem microbiology with an adequate sampling: a proposal for standardisation.

Post-mortem microbiology (PMM) is an important tool in forensic pathology, helping to determine the cause and manner of death, especially in difficult scenarios such as sudden unexpected death (SD). Currently, there is a lack of standardization of PMM sampling throughout Europe. We present recommendations elaborated by a panel of European experts aimed to standardize microbiological sampling in the most frequent forensic and clinical post-mortem situations. A network of forensic microbiologists, pathologists and physicians from Spain, England, Belgium, Italy and Turkey shaped a flexible protocol providing minimal requirements for PMM sampling at four practical scenarios: SD, bioterrorism, tissue and cell transplantation (TCT) and paleomicrobiology. Biosafety recommendations were also included. SD was categorized into four subgroups according to the age of the deceased and circumstances at autopsy: (1) included SD in infancy and childhood (0-16 years); (2) corresponded to SD in the young (17-35 years); (3) comprised SD at any age with clinical symptoms; and (4) included traumatic/iatrogenic SD. For each subgroup, a minimum set of samples and general recommendations for microbiological analyses were established. Sampling recommendations for main bioterrorism scenarios were provided. In the TCT setting, the Belgian sampling protocol was presented as an example. Finally, regarding paleomicrobiology, the sampling selection for different types of human remains was reviewed. This proposal for standardization in the sampling constitutes the first step towards a consensus in PMM procedures. In addition, the protocol flexibility to adapt the sampling to the clinical scenario and specific forensic findings adds a cost-benefit value.

Performance of different culture methods and of a commercial molecular assay for the detection of carbapenemase-producing Enterobacteriaceae in nursing homes and rehabilitation centers.

Over the last several years, carbapenemase-producing Enterobacteriaceae (CPE) have been increasingly detected not only among patients in acute care hospitals, but also in long-term care facilities. In this point prevalence survey, residents from three nursing homes and patients in one rehabilitation center were screened for asymptomatic intestinal carriage of CPE by rectal swabs. The first objective was to evaluate the hypothesis of the establishment of a CPE reservoir in a geriatric/chronic care population. Secondly, we evaluated the comparative performances of different culture methods (chromID(®) CARBA, chromID(®) OXA-48, MacConkey with temocillin/meropenem, ertapenem enrichment broth) and a commercial molecular assay (Check-Direct CPE). From the 257 included residents, only one had evidence for CPE carriage. From the rectal swabs of this resident, an OXA-48-producing Klebsiella pneumoniae could be isolated and was confirmed by a molecular assay both on the strain and on the rectal swab. The specificity of the different culture methods and Check-Direct CPE was at least 97 %. Neither enrichment broth nor prolonged incubation up to 48 h increased the yield of CPE. This point prevalence survey shows a low CPE prevalence of 0.39 %. Larger scaled studies are needed in order to confirm the role of chronic care settings as secondary CPE reservoirs and to adjust the infection control and prevention recommendations.

Cronobacter sakazakii bacteremia in a heart transplant patient with polycystic kidney disease.

Infections with Cronobacter sakazakii are mainly described among neonates and infants, with contaminated powdered infant formulas most often incriminated as the cause. We describe here a case of C. sakazakii bacteremia secondary to a suspected cyst infection in a heart-and-kidney transplant patient with polycystic kidney disease.

Population pharmacokinetic modelling of total and unbound cefazolin plasma concentrations as a guide for dosing in preterm and term neonates.

OBJECTIVES: Cefazolin is frequently administered for antimicrobial prophylaxis and treatment of infections. In neonates, pharmacokinetic observations are limited and dosing regimens variable. The aim of this study was to describe the pharmacokinetics of cefazolin in neonates based on total and unbound concentrations to optimize cefazolin dosing. METHODS: Thirty-six neonates [median birth body weight 2720 (range 540-4200) g, current body weight (cBW) 2755 (830-4200) g and postnatal age (PNA) 9 (1-30) days] receiving intravenous cefazolin (50 mg/kg/8 h) were included. Based on 119 total and unbound plasma concentrations, a population pharmacokinetic analysis with a covariate analysis was performed. Monte Carlo simulations were performed aiming for unbound concentrations above an MIC of 8 mg/L (>60% of the time) in all patients. RESULTS: A one-compartment pharmacokinetic model was developed in which total and unbound concentrations were linked by maximum protein binding (Bmax) of 136 mg/L and a dissociation constant (KD) for cefazolin protein binding of 46.5 mg/L. cBW was identified as covariate for volume of distribution (V), bBW and PNA for clearance and albumin plasma concentration for Bmax, explaining 50%, 58% and 41% of inter-individual variability in V, clearance and Bmax, respectively. Based on Monte Carlo simulations, a body weight- and PNA-adapted dosing regimen that resulted in similar exposure across different weight and age groups was proposed. CONCLUSIONS: A neonatal pharmacokinetic model taking into account total and unbound cefazolin concentrations with saturable plasma protein binding was identified. As cBW and PNA were the most important covariates, these may be used for individualized dosing in neonates.

Shedding a light on ultraviolet-C technologies in the hospital environment.

Ultraviolet (UV)-C light for disinfection has experienced a surge in popularity since the outbreak of COVID-19. Currently, many different UV-C systems, with varied properties that impact disinfection performance, are available on the market. Therefore this review aims to bundle the available information on UV-C disinfection to obtain an overview of its advantages, disadvantages, and performance-influencing parameters. A literature search was performed using the snowball search method in Google Scholar and PubMed with the following keywords: UV-C disinfection, UV-C dose, UV-C light source, UV-C repair mechanism, UV-C photoreactivation, and UV-C disinfection standards. The main parameters of UV-C disinfection are wavelength, dose, relative humidity, and temperature. There is no consensus about their optimal values, but, in general, light at a high dose and a spectrum of wavelengths containing 260 nm is preferred in an environment at room temperature with low relative humidity. This light can be generated by mercury-vapour, light-emitting diode (LED), pulsed-xenon, or excimer lamps. Multiple factors are detrimental to disinfection performance such as shadowing, a rough surface topography, a high level of contamination, repair mechanisms, and the lack of standardization. Also, there are health and safety risks associated with the UV-C technology when used in the proximity of people. UV-C disinfection systems have promising features and the potential to improve in the future. However, clarifications surrounding the different parameters influencing the technologies' effectiveness in hospital environment are needed. Therefore UV-C disinfection should currently be considered for low-level rather than high-level disinfection.

Clinical evaluation of the Copan ESwab for methicillin-resistant Staphylococcus aureus detection and culture of wounds.

The screening for and diagnosis of bacteriological infections often involves the collection and transportation of swab samples. The Copan ESwab was compared with the dry cotton Copan swab for methicillin-resistant Staphylococcus aureus (MRSA) screening (n = 200 paired samples) and with the Amies agar gel swab (Copan) for the sampling of burn and orthopaedic wounds (n = 203 paired samples) in terms of Gram staining and bacterial recovery. Gram stains performed with ESwab liquid showed significantly more Gram-negative rods, streptococci, Gram-positive cocci, Gram-positive rods, polymorphonuclear cells, lymphocytes and red blood cells than Gram stains from dry swabs. Bacterial recovery was significantly higher with ESwab (p < 0.01, for both MRSA screening and wounds, quantitative/semi-quantitative method). This lead to a slightly higher detection rate of MRSA (128 vs. 124 MRSA-positive ESwabs and dry swabs, respectively, p = 0.50) and a higher detection rate of coagulase-negative Staphylococcus spp. (44 isolates with ESwab vs. 29 with Amies gel swab, p = 0.001) and Enterococcus spp. (15 isolates with ESwab vs. 7 isolates with Amies gel swab, p = 0.005) with ESwab (quantitative method). We confirmed that ESwab has a high performance for Gram stains and a higher bacterial recovery than dry and Amies gel swabs when using clinical samples for MRSA screening and wound sampling.

Epidemiology of mucormycosis: review of 18 cases in a tertiary care hospital.

Mucormycosis is an angio-invasive mycosis with high morbidity and mortality rates which mainly affects immunocompromised patients. It is no longer an uncommon disease due to the increased incidence of diabetes and use of immunosuppressive agents in the current era. Our objective was to review all cases of proven and probable mucormycosis--according to EORTC criteria--diagnosed from 2000 until 2007 at the University Hospitals Leuven, a 1900-bed tertiary care hospital, to assess the changing epidemiology of the disease. In 45 patients there was microbiological or histopathological evidence for the presence of a member of Mucorales during the hospital stay of which 12 cases fulfilled the criteria for proven mucormycosis and 6 for probable mucormycosis. The overall incidence was 0.042 cases per 10,000 patient days. A slight although not statistically significant increase in incidence was noticeable during the study period. The major site of infection was the lungs (78% of the cases), with haematological malignancy the most common underlying disorder and Rhizopus species the most often suspected etiologic agent. Overall mortality was 55% and co-infections with Aspergillus species, proven or probable, noted in 44% of cases. The highest survival rate was achieved with surgery combined with antifungal therapy.

Influence of postmortem time on the outcome of blood cultures among cadaveric tissue donors.

Tissue banks provide tissues of human cadaver donors for transplantation. The maximal time limit for tissue retrieval has been set at 24 h postmortem. This study aimed at evaluating the evidence for this limit from a microbiological point of view. The delay of growth in postmortem blood cultures, the identification of the species isolated and clinical/environmental factors were investigated among 100 potential tissue donors. No significant difference was found in the rate of donors with grown blood cultures within (25/65=38%) compared with after (24/65=37%) 24 h of death. Coagulase-negative staphylococci and gastro-intestinal microorganisms were isolated within and after 24 h of death. Two factors--antimicrobial therapy and "delay before body cooling"--were significantly inversely related with donors' blood culture results. From a microbiological point of view, there is no evidence for avoiding tissue retrieval among donors after 24 h of death.

Effectiveness of antibiotics and antiseptics on coagulase-negative staphylococci for the decontamination of bone allografts.

Bone allografts retrieved from multi-organ donors can be decontaminated with minimally aggressive methods. Therefore, we evaluated the efficacy of antibiotics and antiseptics in the decontamination of bone fragments actively contaminated with coagulase-negative staphylococci. Gentamicin (512/1,024 microg/mL), rifampicin (400/1,000 microg/mL), chlorhexidine in alcohol and chlorhexidine soap were tested with different contact times and temperatures and a delay in starting decontamination. Gentamicin-susceptible strains dried on bone could be removed by gentamicin 512 microg/mL after 19 h of contact, while strains not dried on bone could be eliminated by soaking bone for 60 min in gentamicin 512 microg/mL. Rifampicin-susceptible strains could be eliminated by soaking bone for 60 min in rifampicin 1,000 microg/mL. In none of the experimental conditions could gentamicin/rifampicin-resistant staphylococci be eliminated. Antiseptics could not eliminate staphylococci from bone. Different antibiotics need different protocols in order to decontaminate bone allografts.

Post-mortem microbiology in sudden death: sampling protocols proposed in different clinical settings.

BACKGROUND: Autopsies, including minimally invasive autopsies, are a powerful tool for determination of the cause of death. When a patient dies from an infection, microbiology is crucial to identify the causative organism. Post-mortem microbiology (PMM) aims to detect unexpected infections causing sudden deaths; confirm clinically suspected but unproven infection; evaluate the efficacy of antimicrobial therapy; identify emergent pathogens; and recognize medical errors. Additionally, the analysis of the thanatomicrobiome may help to estimate the post-mortem interval. AIMS: The aim was to provide advice in the collection of PMM samples and to propose sampling guidelines for microbiologists advising autopsy pathologists facing different sudden death scenarios. SOURCES: A multidisciplinary team with experts in various fields of microbiology and autopsies on behalf of the ESGFOR (ESCMID - European Society of Clinical Microbiology and Infectious Diseases - study group of forensic and post-mortem microbiology and in collaboration with the European Society of Pathology) developed this narrative review based on a literature search using MedLine and Scopus electronic databases supplemented with their own expertise. CONTENT: These guidelines address measures to prevent sample contamination in autopsy microbiology; general PMM sampling technique; protocols for PMM sampling in different scenarios and using minimally invasive autopsy; and potential use of the evolving post-mortem microbiome to estimate the post-mortem interval. IMPLICATIONS: Adequate sampling is paramount to identify the causative organism. Meaningful interpretation of PMM results requires careful evaluation in the context of clinical history, macroscopic and histological findings. Networking and closer collaboration among microbiologists and autopsy pathologists is vital to maximize the yield of PMM.

Microbiological diagnostics of bloodstream infections in Europe-an ESGBIES survey.

OBJECTIVES: High-quality diagnosis of bloodstream infections (BSI) is important for successful patient management. As knowledge on current practices of microbiological BSI diagnostics is limited, this project aimed to assess its current state in European microbiological laboratories. METHODS: We performed an online questionnaire-based cross-sectional survey comprising 34 questions on practices of microbiological BSI diagnostics. The ESCMID Study Group for Bloodstream Infections, Endocarditis and Sepsis (ESGBIES) was the primary platform to engage national coordinators who recruited laboratories within their countries. RESULTS: Responses were received from 209 laboratories in 25 European countries. Although 32.5% (68/209) of laboratories only used the classical processing of positive blood cultures (BC), two-thirds applied rapid technologies. Of laboratories that provided data, 42.2% (78/185) were able to start incubating BC in automated BC incubators around-the-clock, and only 13% (25/192) had established a 24-h service to start immediate processing of positive BC. Only 4.7% (9/190) of laboratories validated and transmitted the results of identification and antimicrobial susceptibility testing (AST) of BC pathogens to clinicians 24 h/day. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry from briefly incubated sub-cultures on solid media was the most commonly used approach to rapid pathogen identification from positive BC, and direct disc diffusion was the most common rapid AST method from positive BC. CONCLUSIONS: Laboratories have started to implement novel technologies for rapid identification and AST for positive BC. However, progress is severely compromised by limited operating hours such that current practice of BC diagnostics in Europe complies only partly with the requirements for optimal BSI management.

Bacteriostasis testing on allograft tissue inoculated in Wilkins-Chalgren broth.

Tissue banks culture tissue specimens to confirm the absence of viable micro-organisms after decontamination with antibiotics. It is possible that antibiotic residues attached to decontaminated tissue are introduced into enrichment culture media. These could have an inhibitory effect on the culture results and generate false-negative results. Our aim was to detect bacteriostasis in Wilkins-Chalgren broth inoculated with bone and tendon remnants. These remnants had been soaked in a solution containing gentamicin as part of the tissue-processing procedure. We used the United States Pharmacopeia method for bacteriostasis testing with gentamicin-susceptible Pseudomonas aeruginosa American Type Culture Collection (ATCC) 15442, Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633 as test strains, and gentamicin-resistant Candida albicans ATCC 90029 as control. The residual gentamicin concentration in the broths was determined and gentamicin-soaked tissue was placed on Mueller-Hinton agar inoculated with a staphylococcal suspension. Bacteriostasis was present in 53-75% of the reference test strains. Tendon remnants had a significantly higher rate of bacteriostasis (85%) than bone remnants (28%). Broths inoculated with tendon remnants had the highest residual gentamicin concentrations.